Genomic and proteomic approaches to understand oral cancer [unreadable] [unreadable] Although risk factors for HNSCC, such as alcohol and tobacco consumption, are well recognized, the molecular mechanisms responsible for this malignancy are still not fully understood. We have used a number of novel approaches to investigate gene and protein expression profiles in HNSCC. We have shown that laser capture microdissection (LCM) can be used to procure specific cell populations from heterogenous tumor samples, and that LCM-procured material can be used effectively to extract RNA, DNA, and proteins. We have teamed up with other research institutions to conduct gene and protein expression analysis of HNSCC by combining LCM, gene arrays, and proteomic platforms. These efforts have already provided a wealth of information about the distinctive pattern of gene and protein expression in HNSCC. [unreadable] [unreadable] Proteome-wide analysis of HNSCC tissues using LCM and mass spectrometry. Ultimately, the aberrant expression and activity of molecules present in HNSCC cells are responsible for their malignant behavior. Thus, we have recently made a concerted effort to investigate protein expression profiles during HNSCC tumor development. While in prior studies we have investigated protein expression profiles in frozen HNSCC samples, we now explored the use of standard formalin-fixed paraffin-embedded (FFPE) tissues for proteomic analysis, as they preserve the tissue architecture and may reflect better the protein composition as it exists in vivo. We combined LCM with protein extraction procedures and highly sensitive mass spectrometry techniques to identify proteins expressed in normal oral squamous epithelium and HNSCCs displaying distinct differentiation patterns. Mass spectrometry and bioinformatic analysis led to the identification of large numbers of molecules, including cytokeratins, differentiation markers, and proteins involved in stem cell maintenance, signal transduction, migration, cell cycle regulation, angiogenesis, matrix degradation, and proteins with tumor suppressive and oncogenic potential. Representative proteins were further validated using immunohistochemical studies in HNSCC tissue sections and tissue microarrays. Taken together, the ability to combine LCM and in-depth proteomic analysis of FFPE tissues provided valuable information regarding the nature of the proteins expressed in normal squamous epithelium and during HNSCC progression. [unreadable] [unreadable] [unreadable] Dysregulated signaling networks in HNSCC: novel mechanism-based approaches for HSNCC treatment [unreadable] [unreadable] There is an urgent need for new treatment options for HNSCC patients, considering that their overall 5-year survival is relatively low (50%) and has not improved much over the past 3 decades. The emerging information on the nature of the deregulated molecular mechanisms responsible for HNSCC progression has provided the possibility of exploring new mechanisms-based therapeutic approaches for HNSCC. For example, we have observed that persistent activation of the serine-threonine kinase Akt is a frequent event in HNSCC, and that its blockade inhibits tumor cell growth. Among the many molecules whose activity is regulated by Akt, we have made a focal effort on the mammalian target of rapamycin (mTOR), an atypical kinase involved in energy sensing and growth promoting signaling pathways. We have shown that a high fraction of HNSCC clinical samples exhibit high levels of phosphorylated ribosomal S6 protein (pS6), the most downstream target of the Akt-mTOR pathway, and that inhibition of mTOR by the use of rapamycin causes the rapid decrease in the level of pS6 and the apoptotic death of HNSCC tumor xenografts, thereby causing tumor regression. These efforts have identified the Akt-mTOR pathway as a potential therapeutic target for HNSCC.[unreadable] [unreadable] Tumor cells are the primary target for rapamycin in HNSCC - A retro-inhibition approach. Although blockade of mTOR by rapamycin promotes the rapid regression of HNSCC-tumor xenografts in mice, rapamycin does not affect the growth of HNSCC cells in vitro, thus raising the possibility that, as for other cancer types, rapamycin may not target cancer cells directly but may instead act based on its anti-angiogenic properties. To assess whether the cancer cell-autonomous actions of rapamycin contributes to its antitumoral effects, we utilized a retro-inhibition approach consisting in the expression of a rapamycin-resistant form of mTOR (mTOR-RR) in HNSCC cells. Host-derived endothelial and stromal cells retained wild-type mTOR alleles, hence remaining rapamycin-sensitive. Expression of mTOR-RR prevented the decrease in pS6 levels caused by rapamycin through mTOR in HNSCC cells but not in stromal cells, and rendered HNSCC xenografts resistant to the antitumoral activity of rapamycin. This novel reverse-pharmacology strategy also enabled monitoring the direct consequences of inhibiting mTOR in cancer cells within the complex tumor micro-environment, which revealed that mTOR controls the expression of HIF-1, VEGF and a glucose transporter, Glut-1, in HNSCC cells. These findings indicate that HNSCC cells are the primary target of rapamycin in vivo, and provide evidence that its anti-angiogenic effects may represent a downstream consequence of mTOR inhibition in cancer cells. [unreadable] [unreadable] The Head and Neck Tissue Array Initiative: Dissection of the Akt-mTOR signaling network. As an approach to evaluate the expression pattern and status of activation of signaling pathways in clinical specimens from HNSCC patients, we established the Head and Neck Cancer Tissue Array Initiative, an international consortium aimed at developing a high density oral specific HNSCC tissue microarray. Emerging information by the use of phospho-specific antibodies detecting the activated state of signaling molecules indicates that the Akt/mTOR pathway is frequently activated in HNSCC, but independently from the activation of EGFR or the detection of mutant p53. Indeed, we identified a large group of tissue samples displaying active Akt and mTOR in the absence of EGFR activation. These findings provide important information about the nature of the dysregulated signaling networks in HNSCC, and may also provide the rationale for the future selection of HNSCC patients that may benefit from novel mechanism-based therapies.[unreadable] [unreadable] Animal models for oral malignancies [unreadable] [unreadable] A major limitation in the area of HNSCC research is the limited availability of animal models to test the validity of current genetic paradigms of tumorigenesis, and to explore the effectiveness of treatment modalities or chemopreventive approaches. We have been using our recently developed tetracycline-inducible system to express oncogenes in the skin and oral mucosa and other conditional animal systems to explore the contribution of recently identified dysregulated molecular mechanism to SCC progression. [unreadable] [unreadable] Animal models to study the role of the Akt-mTOR pathway in SCC: The emerging studies supporting a role for the Akt-mTOR pathway in HNSCC development prompted us to direct our efforts, and focus on the development of animal models in which we could conditionally activate this signaling route. Indeed, stimulation of PI3K-Akt-mTOR by the epithelial-specific deletion of Pten, a PIP3 lipid phosphatase, was sufficient to cause multiple hyperproliferative and tumor lesions in the skin and mouth and resulted in early lethality. This suggests that decreased PTEN activity in humans, which is a frequent genetic or epigenetic event in HNSCC, may contribute to tumor development. Remarkably, the prolonged administration of low doses of rapamycin promoted the rapid regression of advanced mucocutaneous lesions, and was sufficient to prevent their development if initiated before disease manifestation, underscoring the key role of mTOR for tumor progression in squamous epithelium.